Transdiscal administration of high affinity anti-MMP inhibitors

ABSTRACT

The present invention relates to injecting a high affinity antagonist of MMPs into a diseased intervertebral disc.

RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/470,098, filed May 13, 2003, and is acontinuation-in-part application of U.S. patent application Ser. No.10/456,948, DiMauro et al., filed Jun. 6, 2003. The entire teachings ofthe above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The natural intervertebral disc contains a jelly-like nucleuspulposus surrounded by a fibrous annulus fibrosus. Under an axial load,the nucleus pulposus compresses and radially transfers that load to theannulus fibrosus. The laminated nature of the annulus fibrosus providesit with a high tensile strength and so allows it to expand radially inresponse to this transferred load.

[0003] In a healthy intervertebral disc, cells within the nucleuspulposus produce an extracellular matrix (ECM) containing a highpercentage of proteoglycans. These proteoglycans contain sulfatedfunctional groups that retain water, thereby providing the nucleuspulposus with its cushioning qualities. These nucleus pulposus cells mayalso secrete small amounts of cytokines as well as matrixmetalloproteinases (MMPs). These cytokines and MMPs help regulate themetabolism of the nucleus pulposus cells.

[0004] In some instances of disc degeneration disease (DDD), gradualdegeneration of the intervertebral disc is caused by mechanicalinstabilities in other portions of the spine. In these instances,increased loads and pressures on the nucleus pulposus cause the cellswithin the disc (or invading macrophages) to emit larger than normalamounts of the above-mentioned cytokines. In other instances of DDD,genetic factors or apoptosis can also cause the cells within the nucleuspulposus to emit toxic amounts of these cytokines and MMPs. In someinstances, the pumping action of the disc may malfunction (due to, forexample, a decrease in the proteoglycan concentration within the nucleuspulposus), thereby retarding the flow of nutrients into the disc as wellas the flow of waste products out of the disc. This reduced capacity toeliminate waste may result in the accumulation of high levels of toxinsthat may cause nerve irritation and pain.

[0005] As DDD progresses, toxic levels of the cytokines and MMPs presentin the nucleus pulposus begin to degrade the extracellular matrix. Inparticular, the MMPs (as mediated by the cytokines) begin cleaving thewater-retaining portions of the proteoglycans, thereby reducing itswater-retaining capabilities. This degradation leads to a less flexiblenucleus pulposus, and so changes the loading pattern within the disc,thereby possibly causing delamination of the annulus fibrosus. Thesechanges cause more mechanical instability, thereby causing the cells toemit even more cytokines, typically thereby upregulating MMPs. As thisdestructive cascade continues and DDD further progresses, the discbegins to bulge (“a herniated disc”), and then ultimately ruptures,causing the nucleus pulposus to contact the spinal cord and producepain.

[0006] U.S. Published patent application No. US 2003/0039651 (“OlmarkerI”) teaches a therapeutic treatment of nerve disorders comprisingadministration of a therapeutically effective dosage of compounds,including inhibitors of MMPs.

[0007] In the examples of Olmarker I, it is taught that the therapeuticcompounds are to be administered through systemic pathways. Inparticular, Olmarker I teaches that “the major contribution of TNF-alphamay be derived from recruited, aggregated and maybe even extravasatedleukocytes, and that successful pharmacologic block may be achieved onlyby systemic treatment. (0133). Of note, Olmarker I appears to discouragethe local addition of at least one therapeutic compound (doxycycline) toan autotransplanted nucleus pulposus to be applied to a spinal cord.(0128)

[0008] PCT Published Patent Application No. WO 02/100387 (“Olmarker II”)teaches the prevention of neovascularization and/or neo-innervation ofintervertebral discs by the administration of anti-angiogenicsubstances. Again, however, Olmarker II teaches systemic administrationof these therapeutic agents.

[0009] U.S. Patent No. 6,419,944 (“Tobinick”) discloses treatingherniated discs with cytokine antagonists. Tobinick teaches that localadministration involves a subcutaneous injection near the spinal cord.Accordingly, Tobinick does not teach a procedure involving a sustaineddelivery of a drug for the treatment of DDD, nor directly administeringa drug into the disc.

[0010] U.S. Published patent application No. 2003/0049256 (Tobinick II)discloses that injection of such therapeutic molecules to the anatomicarea adjacent to the spine is accomplished by interspinous injection,and preferably is accomplished by injection through the skin in theanatomic area between two adjacent spinous processes of the vertebralcolumn.

[0011] Tobinick II further teaches that the therapeutic compounds may beadministered by interspinous injection in the human and that the dosagelevel is in the range of 1 mg to 300 mg per dose, with dosage intervalsas short as two days. Tobinick II further discloses that othertherapeutic compounds are administered in a therapeutically effectivedose, which will generally be 10 mg to 200 mg per dose, and their dosageinterval will be as short as once daily.

[0012] Tobinick, Swiss Med. Weekly, 133: 170-7 (2003) (“Tobinick III”)teaches perispinal and epidural administration of TNF inhibitors.

[0013] Karppinen, Spine, 28(8): 750-4 (2003), teaches intravenouslyinjecting or orally administering infliximab into patients sufferingfrom sciatica.

[0014] As with Tobinick I and II, Karppinen does not teach a procedureinvolving a sustained delivery of a drug for the treatment of DDD, nordirectly administering a drug into the disc space.

[0015] U.S. Pat. No. 6,352,557 (Ferree) teaches adding therapeuticsubstances such as anti-inflammatory medications to morselizedextra-cellular matrix, and injecting that combination into anintervertebral disc. However many anti-inflammatory agents arenon-specific and therefore may produce unwanted side effects upon othercells, proteins and tissue. In addition, the pain-reducing effect ofthese agents is typically only temporary. Lastly, these agents typicallyonly relieve pain, and are neither curative nor restorative.

[0016] Alini, Eur. Spine J., 11(Supp. 2): S215-220 (2002), teachestherapies for early stage DDD, including injection of inhibitors ofproteolytic enzymes or biological factors that stimulate cell metabolicactivity (i.e., growth factors) in order to slow down the degenerativeprocess. Inhibitors of proteolytic enzymes constitute a broad class ofcompounds, including i) inhibitors of proteolytic enzyme synthesis andii) inhibitors of proteolytic enzyme activity. Alini I does not specifyany desired types of inhibitors of proteolytic enzymes.

[0017] U.S. Published patent application US 2002/0026244 (“Trieu”)discloses an intervertebral disc nucleus comprising a hydrogel that maydeliver desired pharmacological agents. Trieu teaches that thesepharmacological agents may include growth factors such as TGF-β andanti-inflammatory drugs, including steroids. Trieu further teaches thatthese pharmacological agents may be dispersed within the hydrogel havingan appropriate level of porosity to release the pharmacological agent ata desired rate. Trieu teaches that these agents may be released uponcyclic loading or upon resorption.

[0018] Goupille, Spine, 23(14): 1612-1626 (1998) identifies TissueInhibitors of MMPs (“TIMPs”) as degraders of MMP activity. Goupillereports that TIMP-1 and TIMP-2 bind noncovalently to active MMPs in a1:1 molar ratio and specifically inhibit their enzymatic activity.However, Goupille also indentifies corticosteroids, retinoic acid,TGF-B, PGE1 and PGE2 as inhibitors of MMP synthesis; identifiesα2-macroglobulin, hydroxamic acid, derivatives, tetracyclines andquinolones as inhibitors of MMP activity, and identifies bFGF, EGF,Retenoic acid, TGF-β, IL-6, IL-1 LIF, dexamethasone, phorbol ester, andsynthetic Vitamin A analogs as stimulators of TIMPs. Moreover, as toadministration route, Goupille explicitly identifies only the oraladministration route.

SUMMARY OF THE INVENTION

[0019] The present inventors have developed a number of procedures forefficaciously treating degenerative disc disease by drug therapy.

[0020] In accordance with the present invention, the present inventorshave developed a method of treating an intervertebral disc in which ahigh affinity anti-MMP compound (“HAAMMP”) is administered transdiscally(directly into a degenerating disc).

[0021] There are believed to be several advantages to directlytransdiscally administering HAAMMP to a targeted disc:

[0022] First, HAAMMPs inhibit the activity of MMPs. Since it is knownthat MMPs play primary roles in the degradation of the extracellularmatrix (ECM) of the nucleus pulposus, injecting HAAMMPs directly intothe disc in a therapeutically effective amount can prevent the MMPs fromcausing any further ECM degradation. In effect, the transdiscaladminstration of HAAMMPs helps arrest the aging process of thedegenerating disc. Accordingly, the present invention seeks to treat thedegenerative disc at a much earlier stage of DDD and thereby preventsdegradation of the ECM.

[0023] Second, since the HAAMMP is specific, it does not inhibitnon-targeted cells, tissue or proteins. In addition, the HAAMMP may becombined with other therapeutic agents (such as growth factors ormesenchymal stem cells) that can also be injected into the disc withoutreducing the effectiveness of those agents.

[0024] Third, since the annulus fibrosus portion of the disc comprises adense fibrosus structure, this outer component of the disc may provide asuitable depot for the HAAMMP, thereby increasing its half-life in thedisc.

[0025] Accordingly, in one aspect of the present invention, there isprovided a method of treating degenerative disc disease in anintervertebral disc having a nucleus pulposus, comprising transdiscallyadministering a formulation comprising an effective amount of a highaffinity anti-matrix metalloproteinase (HAAMMP) into an intervertebraldisc.

[0026] In some embodiments, the HAAMMP is administered in a formulationcomprising a sustained release device.

DETAILED DESCRIPTION OF THE INVENTION

[0027] For the purposes of the present invention, the terms “inhibitor”and antagonist” are used interchangeably. A protein may be inhibited atthe synthesis level, at the translation level, by shedding, byantibodies, or by soluble receptors. The term “patient” refers to ahuman having a degenerating disc.

[0028] For the purposes of the present invention “transdiscaladministration” includes, but is not limited to:

[0029] a) injecting a formulation into the nucleus pulposus of adegenerating disc, such as a relatively intact degenerating disc;

[0030] b) injecting a formulation into the annulus fibrosus of adegenerating disc, such as a relatively intact degenerating disc;

[0031] c) providing a formulation in a patch attached to an outer wallof the annulus fibrosus;

[0032] d) providing a formulation in a depot at a location outside butclosely adjacent to an outer wall of the annulus fibrosus(“trans-annular administration”); and

[0033] e) providing a formulation in a depot at a location outside butclosely adjacent to an endplate of an adjacent vertebral body(hereinafter, “trans-endplate administration”.

[0034] Because DDD is a continuous process, the degenerating disc towhich the therapeutic drug is administered may be in any one of a numberof degenerative states. Accordingly, the degenerating disc may be anintact disc. The degenerating disc may be a herniated disc (i.e.,wherein a portion of the annulus fibrosus has a bulge). The degeneratingdisc may be a ruptured disc (i.e., wherein the annulus fibrosus hasruptured and the bulk nucleus pulposus has exuded). The degeneratingdisc may be delaminated (i.e., wherein adjacent layers of the annulusfibrosus have separated). The degenerating disc may have fissures (i.e.,wherein the annulus fibrosus has fine cracks or tears through whichselected molecules from the nucleus pulposus can leak).

[0035] The present invention is directed to providing a HAAMMP to adiseased intervertebral disc. In one embodiment, the HAAMMP isadministered in an amount effective to inhibit the specific action ofMMPs released by disc cells during the degenerative process. In oneembodiment, the HAAMMP is administered in an amount effective to inhibitMMPs present in the nucleus pulposus and thereby help arrest degradationof an extracellular matrix.

[0036] In one embodiment, the HAAMMP is recombinant. In one embodiment,the HAAMMP is present in an autologous form. The HAAMMP can be present,for example, in an autologous concentrated form.

[0037] In some embodiments, the HAAMMP is a natural inhibitor of MMPs,e.g., a tissue inhibitor of MMPs (TIMP). In some embodiments, the TIMPis selected from the group consisting of TIMP-1 and TIMP-2. In someembodiments, the TIMP is autologous and is concentrated by filtration,centrifugation or by immuno-attachment processes. In other embodiments,the TIMP is manufactured recombinantly, and is preferably present in aconcentration of, e.g., at least 1000 times that found normally in thepatient. In some embodiments, the TIMP can be obtained from aheterologous source.

[0038] In some embodiments, the HAAMMP is a specific inhibitor ofaggrecanase (i.e., anti-aggrecanase).

[0039] In some embodiments, the HAAMMP comprises a chelating group thatbinds tightly to the zinc component present in the active site of theMMP. Such HAAMMPs may be selected from the materials disclosed inGordon, Clin. Exp. Rheumatol., 1(Supp 8): S91-4 (1993); and Johnson, J.,Enzyme Inhib., 2:1-22 (1987).

[0040] In some embodiments, the HAAMMP is a specific antagonist of acollagenase MMP. In some embodiments, the therapeutic substance is aspecific antagonist of a stromelysin MMP. In some embodiments, thetherapeutic substance is a specific antagonist of a gelatinase MMP. Insome embodiments, the therapeutic substance is a specific antagonist ofa membrane MMP.

[0041] In some embodiments, the targeted MMP is selected from the groupconsisting MMP-2, MMP-3 and MMP-8. Targeting MMP-2 and/or MMP-3 isdesirable because these MMPs are believed to degrade proteoglycans.Targeting MMP-8 is desirable because this MMP is believed to degradeaggrecans.

[0042] DDD involves the progressive degeneration of a disc in which manyfactors are involved. In many instances, simply providing a single doseor even a regimen over the space of a few days may not be sufficient toresolve the DDD. For example, if DDD were caused in part by mechanicalinstability in a functional spinal unit, then simply providing aone-time therapy for the transdiscal cells would likely only delay theonset of the DDD. Therefore, there is a need to provide a long-term drugtherapy treatment of DDD that does not require multiple injections.

[0043] Because DDD is a continuous process, it is desirable for theHAAMMP to remain within the nucleus pulposus as long as possible in apharmaceutically effective amount. The half-life of the HAAMMP withinthe nucleus pulposus will depend upon many factors, including the sizeof the HAAMMP and its charge. In general, the larger the molecularweight of the HAAMMP, the more likely it is to remain contained by theannulus fibrosus portion of the disc.

[0044] When using a short half-life HAAMMP, it would be desirable for arelatively large dose of the HAAMMP to be administered transdiscally. Inthis condition, quick depletion of the HAAMMP would not cause the HAAMMPto fall below therapeutically effective levels in the disc until anextended period.

[0045] Although a large dose of the HAAMMP would be desirable in suchinstances, injecting a critical volume of water can increase pressure inthe nucleus pulposus. Nociceptors present on the inner wall of theannulus fibrosus react to this increased pressure and produce pain. Insome cases, the added amount could be as little as one cc by volume toproduce pain. Accordingly, if a dilute concentration of a HAAMMP isadded to the nucleus pulposus to provide a large dose, the resultingpressure increase caused by this added volume could be sufficient tocause acute pain.

[0046] For example, if it were determined that about 100 mg of a HAAMMPwas needed to therapeutically affect a nucleus pulposus, and that HAAMMPwas provided in concentrations of from about 30 to about 60 mg/ml, thenat least about 1.5 ml of the HAAMMP would need to be injected into thenucleus pulposus in order to provide the desired therapeutic effect.However, when injecting volumes into the nucleus pulposus, it isdesirable that the volume of drug delivered be no more than about 1 ml,preferably no more than about 0.5 ml (i.e., a maximum of 0.5 ml.), morepreferably between about 0.1 and about 0.3 ml. When injected in thesesmaller quantities, it is believed the added volume will not cause anappreciable pressure increase in the nucleus pulposus.

[0047] Accordingly, in some embodiments, the concentration of the HAAMMPin the administered formulation is at least about 100 mg/ml. When about100 mg of the HAAMMP is needed to produce the desired therapeuticresult, no more than about 1 ml of the drug need be injected. In someembodiments, the concentration of the HAAMMP in the administered drug isat least about 200 mg/ml. In this condition, no more than about 0.5 mlof the drug need be injected. In some embodiments, the concentration ofthe HAAMMP in the administered drug is at least about 500 mg/ml. In thiscondition, between about 0.03 to about 0.3 ml of the formulation need beinjected.

[0048] In some embodiments, the HAAMMP is provided in a sustainedrelease device (i.e., sustained delivery device). The sustained releasedevice is adapted to remain within the disc for a prolonged period andslowly release the HAAMMP contained therein to the surroundingenvironment. This mode of delivery allows a HAAMMP to remain intherapeutically effective amounts within the disc for a prolongedperiod.

[0049] In some embodiments, the HAAMMP is predominantly released fromthe sustained delivery device by its diffusion through the sustaineddelivery device (e.g., through a polymer). In others, the HAAMMP ispredominantly released from the sustained delivery device by thebiodegradation of the sustained delivery device (e.g., biodegradation ofa polymer).

[0050] In some embodiments, the sustained release device comprises abioresorbable material whose gradual erosion causes the gradual releaseof the HAAMMP to the disc environment. In some embodiments, thesustained release device comprises a bioresorbable polymer. In someembodiments, the bioresorbable polymer has a half-life of at least onemonth, more preferably at least two months, more preferably at least 6months.

[0051] In some embodiments, the sustained delivery device comprisesbioerodable macrospheres. The HAAMMP is preferably contained in agelatin (or water or other solvent) within the capsule, and is releasedto the disc environment when the outer shell has been eroded. The devicecan include a plurality of capsules having outer shells of varyingthickness, so that the sequential breakdown of the outer shells providesperiodic release of the HAAMMP.

[0052] In some embodiments, the sustained release device providescontrolled release. In some embodiments, it provides continuous release.In some embodiments, it provides intermittent release. In someembodiments, the sustained release device comprises a biosensor. Otherrelease modes may also be used.

[0053] In some embodiments, the sustained delivery device comprises aninflammatory-responsive delivery system, such as one comprisingbioerodable microspheres that are eroded by invading macrophages. Thistechnology provides a high correspondence between physiologicinflammation of disc environment and the release of the HAAMMPs intothat environment. In one embodiment, the technology disclosed in Brownet al., Arthritis. Rheum. Dec., 41(12): 2185-95 (1998) is selected.

[0054] In some embodiments, the sustained delivery device comprises thedevices disclosed in U.S. Pat. No. 5,728,396 (“Peery”), thespecification of which is incorporated by reference in its entirety.

[0055] In some embodiments, the sustained delivery device comprises aplurality (e.g., at least one hundred) of water-containing chambers,each chamber containing the HAAMMP. A chamber is defined by bilayerlipid membranes comprising synthetic duplicates of naturally occurringlipids. Release of the drug can be controlled by varying at least one ofthe aqueous excipients, the lipid components, and the manufacturingparameters. Preferably, the formulation comprises no more than 10%lipid. In some embodiments, the Depofoam™ technology of Skyepharma PLC(London, United Kingdom) is selected.

[0056] In some embodiments, the sustained delivery device comprises adelivery system disclosed in U.S. Pat. No. 5,270,300 (“Hunziker”), thespecification of which is incorporated by reference in its entirety.

[0057] In some embodiments, the sustained delivery device comprises theco-polymer poly-DL-lactide-co-glycolide (PLG). Preferably, theformulation is manufactured by combining the HAAMMP or additionaltherapeutic agent, the co-polymer and a solvent to form a droplet, andthen evaporating the solvent to form a microsphere. The plurality ofmicrospheres are then combined in a biocompatible diluent. Preferably,the HAAMMP or additional therapeutic agent is released from theco-polymer by its diffusion therethrough and by the biodegradation ofthe co-polymer. In some embodiments hereof, the ProLeaseTM technology ofAlkermes (located in Cambridge, Mass.) is selected.

[0058] In some preferred embodiments, the HAAMMP is combined in theformulation with a sustained release device comprising aviscosupplement. The viscosupplement has a viscosity and elasticitysubstantially similar to that of natural healthy nucleus pulposus.

[0059] Preferably, the viscosupplement is selected from the groupconsisting of hyaluronic acid and hyaluronate (either cross-linked oruncross-linked). In some embodiments, the viscosupplement is Arthrease™(DePuy Ltd., Leeds, U.K.). In some embodiments, the viscosupplement is ahyaluronic acid selected from the hyaluronic acids disclosed in U.S.Ser. No. 09/298,539, entitled “Method of Treating Diseased, Injured orAbnormal Cartilage with Hyaluronic Acid and Growth Factors” (Radomsky etal.), the specification of which is incorporated by reference in itsentirety.

[0060] Hydrogels can also be used to deliver the HAAMMP in a sustainedrelease manner to the disc environment. A “hydrogel” is a substanceformed when an organic polymer (natural or synthetic) is set orsolidified to create a three-dimensional open-lattice structure thatentraps molecules of water or other solution to form a gel. Thesolidification can occur, e.g., by aggregation, coagulation, hydrophobicinteractions, or cross-linking. The hydrogels employed in this inventioncan rapidly solidify to keep the HAAMMP at the application site, therebyeliminating undesired migration from the disc. The hydrogels are alsobiocompatible, e.g., not toxic, to cells suspended in the hydrogel.

[0061] A “hydrogel-HAAMMP composition” is a suspension of a hydrogelcontaining HAAMMP. The hydrogel-HAAMMP composition forms a uniformdistribution of HAAMMP with a well-defined and precisely controllabledensity. Moreover, the hydrogel can support very large densities ofHAAMMP. In addition, the hydrogel allows diffusion of nutrients to, andwaste products away from, the endplates, which promotes tissue growth.

[0062] Hydrogels suitable for use in the present invention includewater-containing gels, i.e., polymers characterized by hydrophilicityand insolubility in water. See, for instance, “Hydrogels”, pages 458-459in Concise Encyclopedia of Polymer Science and Engineering, Eds. Mark etal., Wiley and Sons (1990), the disclosure of which is incorporatedherein by reference in its entirety. Although their use is optional inthe present invention, the inclusion of hydrogels is can be highlyadvantageous since they tend to possess a number of desirable qualities.By virtue of their hydrophilic, water-containing nature, hydrogels can:

[0063] a) house viable cells, such as mesenchymal stems cells, and

[0064] b) assist with load bearing capabilities of the disc.

[0065] In one embodiment, the hydrogel is a fine, powdery synthetichydrogel. Suitable hydrogels exhibit an optimal combination of suchproperties as compatibility with the matrix polymer of choice, andbiocompatability. The hydrogel can include any one or more of thefollowing: polysaccharides, proteins, polyphosphazenes,poly(oxyethylene)-poly(oxyproplene) block polymers,poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine,poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acidand methacrylic acid, poly(vinyl acetate), and sulfonated polymers.

[0066] In general, these polymers are at least partially soluble inaqueous solutions, e.g., water, or aqueous alcohol solutions that havecharged side groups, or a monovalent ionic salt thereof. There are manyexamples of polymers with acidic side groups that can be reacted withcations, e.g., poly(phosphazenes), poly(acrylic acids), andpoly(methacrylic acids). Examples of acidic groups include carboxylicacid groups, sulfonic acid groups, and halogenated (preferablyfluorinated) alcohol groups. Examples of polymers with basic side groupsthat can react with anions are poly(vinyl amines), poly(vinyl pyridine),and poly(vinyl imidazole).

[0067] In some embodiments, the sustained delivery device includes apolymer selected from the group consisting of PLA, PGA, PCL and mixturesthereof.

[0068] When using a relatively long-half life HAAMMP, a relatively smalldose of the HAAMMP can be administered into the disc. In this condition,the slow depletion of the HAAMMP would not cause the HAAMMP to fallbelow therapeutically effective levels in the disc until an extendedperiod of time has elapsed.

[0069] In some embodiments in which HAAMMP have long half-lives withinthe disc space, the dose administered can be very small.

[0070] For example, if it is believed that an HAAMMP is effective whenpresent in the range of about 1 to about 10 mg/kg or from about 1 toabout 10 ppm, and since a typical nucleus pulposus of a disc has avolume of about 3 ml (or 3 cc, or 3 g), then only about 3 to about 30 ugof the HAAMMP need be administered to the disc in order to provide along lasting effective amount of the HAAMMP. The formulation can beadministered in an amount of less than 1 cc. As a point of reference,Tobinick discloses that at least 1 mg of cytokine antagonist should beadministered perispinally in order to cure back pain. The smalleramounts available by this route reduce the chances of deleterious sideeffects of the HAAMMP.

[0071] For example, if a clinician administered 0.3 ml of 60 mg/mlHAAMMP into a 2.7 cc disc, this would produce a HAAMMP concentration inthe disc of about 6 mg/ml, or 6 parts per thousand. Without wishing tobe tied to a theory, if HAAMMP has the same half-life within a nucleuspulposus as it does when administered systemically (i.e, about 1 week),then the concentration of HAAMMP would remain above about 10 ppm forabout 9 weeks. Therefore, if another dose were needed, the clinicianwould only need to provide the second dose after about two months.

[0072] Therefore, in some embodiments, the HAAMMP is provided in a doseof less than about 1 mg, e.g., less than about 0.5 mg, more preferably,less than about 0.1 mg, more preferably less than about 0.01 mg, morepreferably less than about 0.001 mg. The smaller amounts available bythis route reduce the chances of deleterious side effects of the HAAMMP.

[0073] In some embodiments, the formulation of the present invention isadministered directly into the disc through the outer wall of theannulus fibrosus. More preferably, the direct administration includesdepositing the HAAMMP in the nucleus pulposus portion of the disc. Inthis condition, the fibrous nature of the annulus fibrosus thatsurrounds the nucleus pulposus will help keep the HAAMMP containedwithin the disc.

[0074] In some embodiments, the formulation of the present invention isinjected into the disc through a small bore needle. In some embodiments,the needle has a bore of about 22 gauge or less, so that thepossibilities of producing a herniation are mitigated. For example, theneedle can have a bore of about 24 gauge or less, so that thepossibilities of producing a herniation are even further mitigated.

[0075] If the volume of the direct injection of the formulation issufficiently high so as to cause a concern of overpressurizing thenucleus pulposus, then it is preferred that at least a portion of thenucleus pulposus be removed prior to direct injection. In someembodiments, the volume of removed nucleus pulposus is substantiallysimilar to the volume of the formulation to be injected. For example,the volume of removed nucleus pulposus can be within 80-120% of thevolume of the formulation to be injected. In addition, this procedurehas the added benefit of at least partially removing some degenerateddisc from the patient.

[0076] In other embodiments, the formulation is delivered into the discspace through the endplate of an opposing vertebral body. This avenueeliminates the need to puncture the annulus fibrosus, and so eliminatesthe possibility of herniation.

[0077] Although the HAAMMP may therapeutically treat the disc by bindingan MMP, thereby reducing pain and arresting degradation of the ECM, itis believed that at least some of these HAAMMPs do not help repair thedamage done by the MMP to the ECM.

[0078] Therefore, there may be a need to provide a therapy that alsohelps repair the ECM.

[0079] In accordance with the present invention, there is provided amethod of treating degenerative disc disease in an intervertebral dischaving a nucleus pulposus, comprising:

[0080] a) transdiscally administering HAAMMP into a degenerating disc;and

[0081] b) transdiscally administering at least one additionaltherapeutic agent in an amount effective to at least partially repairthe disc (i.e., the disc tissue).

[0082] In accordance with one aspect of the invention, both the HAAMMPand at least one additional therapeutic agent are locally administeredinto the disc space. There can be, for example, one additionaltherapeutic agent (i.e., a second therapeutic agent) or there can bemultiple additional therapeutic agents (e.g., second and thirdtherapeutic agents).

[0083] In some embodiments, the HAAMMP and additional therapeutic agent(i.e., additional therapeutic substance) are administeredsimultaneously. In others, the HAAMMP is administered first. In stillother, the second therapeutic agent is administered first.

[0084] Other compounds which may be added to the disc include, but arenot limited to: vitamins and other nutritional supplements; hormones;glycoproteins; fibronectin; peptides and proteins; carbohydrates (bothsimple and/or complex); proteoglycans; oligonucleotides (sense and/orantisense DNA and/or RNA); bone morphogenetic proteins (BMPs);antibodies (for example, to infectious agents, tumors, drugs orhormones); gene therapy reagents; and anti-cancer agents. Geneticallyaltered cells and/or other cells may also be included in the matrix ofthis invention. If desired, substances such as pain killers andnarcotics may also be admixed with the polymer for delivery and releaseto the disc space.

[0085] Healthy cells can be introduced into the disc that can at leastpartially repair any damage done to the disc during the degenerativeprocess. In some embodiments, these cells are introduced into thenucleus pulposus and ultimately produce new extracellular matrix for thenucleus pulposus. In others, these cells are introduced into the annulusfibrosus and produce new extracellular matrix for the annulus fibrosus.

[0086] In some embodiments, these cells are obtained from another humanindividual (allograft), while in other embodiments, the cells areobtained from the same individual (autograft). In some embodiments, thecells are taken from an intervertebral disc (for example, either nucleuspulposus cells or annulus fibrosus cells), while in other embodiments,the cells are taken from a non-disc tissue (for example, mesenchymalstem cells or chondrocytes). In other embodiments, autograftchondrocytes (such as from the knee, hip, shoulder, finger or ear) maybe used.

[0087] In one embodiment, when viable cells are selected as theadditional therapeutic substance, the viable cells comprise mesenchymalstem cells (MSCs). MSCs provide a special advantage for administrationinto a degenerating disc because it is believed that they can morereadily survive the relatively harsh environment present in thedegenerating disc; that they have a desirable level of plasticity; andthat they have the ability to proliferate and differentiate into thedesired cells.

[0088] In some embodiments, the mesenchymal stem cells are obtained frombone marrow, preferably autologous bone marrow. In others, themesenchymal stem cells are obtained from adipose tissue, preferablyautologous adipose tissue.

[0089] In some embodiments, the mesenchymal stem cells injected into thedisc are provided in an unconcentrated form. In other embodiments, theyare provided in a concentrated form. When provided in concentrated form,they can be uncultured. Uncultured, concentrated MSCs can be readilyobtained by centrifugation, filtration, or immunoabsorption. Whenfiltration is selected, the methods disclosed in U.S. Pat. No. 6,049,026(“Muschler”), the specification of which is incorporated by reference inits entirety, are preferably used. In some embodiments, the matrix usedto filter and concentrate the MSCs is also administered into the nucleuspulposus. If this matrix has suitable mechanical properties, it can beused to restore the height of the disc space that was lost during thedegradation process.

[0090] In some embodiments, growth factors are additional therapeuticagents. As used herein, the term “growth factors” encompasses anycellular product that modulates the growth or differentiation of othercells, particularly connective tissue progenitor cells. The growthfactors that may be used in accordance with the present inventioninclude, but are not limited to, members of the fibroblast growth factorfamily, including acidic and basic fibroblast growth factor (FGF-1 and-2) and FGF-4, members of the platelet-derived growth factor (PDGF)family, including PDGF-AB, PDGF-BB and PDGF-AA; EGFs, members of theinsulin-like growth factor (IGF) family, including IGF-I and -II; theTGF-β superfamily, including TGF-β1, 2 and 3 (including MP-52),osteoid-inducing factor (OIF), angiogenin(s), endothelins, hepatocytegrowth factor and keratinocyte growth factor; members of the bonemorphogenetic proteins (BMPs) BMP-1; BMP-3; BMP-2; OP-1; BMP-2A, -2B,-4, -7 and -14; HBGF-1 and HBGF-2; growth differentiation factors(GDFs), members of the hedgehog family of proteins, including indian,sonic and desert hedgehog; ADMP-1; GDF-5; and members of thecolony-stimulating factor (CSF) family, including CSF-1, G-CSF, andGM-CSF; and isoforms thereof.

[0091] In some embodiments, the growth factor is selected from the groupconsisting of TGF-β, bFGF, and IGF-1. These growth factors are believedto promote regeneration of the nucleus pulposus, or stimulateproliferation and/or differentiation of chondrocytes, as well as ECMsecretion. In one embodiment, the growth factor is TGF-β. Morepreferably, TGF-β is administered in an amount of between about 10 ng/mland about 5000 ng/ml, for example, between about 50 ng/ml and about 500ng/ml, e.g., between about 100 ng/ml and about 300 ng/ml.

[0092] In some embodiments, platelet concentrate is provided as anadditional therapeutic agent. In one embodiment, the growth factorsreleased by the platelets are present in an amount at least two-fold(e.g., four-fold) greater than the amount found in the blood from whichthe platelets were taken. In some embodiments, the platelet concentrateis autologous. In some embodiments, the platelet concentrate is plateletrich plasma (PRP). PRP is advantageous because it contains growthfactors that can restimulate the growth of the ECM, and because itsfibrin matrix provides a suitable scaffold for new tissue growth.

[0093] In some embodiments, an additional therapeutic substance is ahigh specificity cytokine antagonist (“HSCA”). For example, the highaffinity cytokine antagonist is selected from the group consisting of anHSCA of TNF-α, and an HSCA of an interleukin.

[0094] In some embodiments, the HSCA is a specific antagonist of TNF-α.Preferred TNF antagonists include, but are not limited to, thefollowing: etanercept (Enbrel® Amgen); infliximab (Remicade® Johnson andJohnson); D2E7, a human anti-TNF monoclonal antibody (KnollPharmaceuticals, Abbott Laboratories); CDP 571 (a humanized anti-TNFIgG4 antibody); CDP 870 (an anti-TNF alpha humanized monoclonal antibodyfragment), both from Celltech; soluble TNF receptor Type I (Amgen);pegylated soluble TNF receptor Type I (PEGs TNF-R1) (Amgen); andonercept, a recombinant TNF binding protein (r-TBP-1) (Serono).

[0095] In some embodiments, the HSCA is a specific antagonist of aninterleukin. Preferably, the target interleukin is selected from thegroup consisting IL-1, IL-2, IL-6 and IL-8, and IL-12. Preferredantagonists include but are not limited to Kineretg (recombinant IL1-RA, Amgen), IL1-Receptor Type 2 (Amgen) and IL-1 Trap (Regeneron).

[0096] Since many pro-inflammatory proteins play a role in discdegeneration, and that the antagonists of the present invention arehighly specific, it is further believed that injecting at least two ofthe highly specific antagonists of the present invention directly intothe disc would be advantageous.

[0097] Therefore, in accordance with the present invention, there isprovide a method of treating degenerative disc disease in anintervertebral disc having a nucleus pulposus, comprising administeringa formulation comprising HAANMP and at least two highly specificantagonists of pro-inflammatory cytokines selected from the groupconsisting of TNF-α, an interleukin (preferably, IL-1, IL-6 and IL-8),FAS, an FAS ligand, and IFN-gamma.

[0098] In one embodiment, at least one of the therapeutic agents is anantagonist of TNF-α. In one embodiment, another therapeutic agent is anantagonist of an interleukin.

[0099] In some embodiments, the formulation comprises a suitablebiocompatible carrier such as saline. In some embodiments, the carrieris selected from the carriers disclosed in U.S. Pat. No. 6,277,969(“Le”), the specification of which is incorporated by reference in itsentirety.

[0100] Also in accordance with the present invention, there is provideda kit comprising a device containing:

[0101] a) a HAAMMP; and

[0102] b) at least one additional therapeutic agent comprising MSCspresent in an amount effective to at least partially repair adegenerating disc.

[0103] Also in accordance with the present invention, there is provideda formulation for treating degenerative disc disease, comprising:

[0104] a) HAAMMP, and

[0105] b) at least one additional therapeutic agent selected from thegroup consisting of:

[0106] i) a growth factor, and

[0107] ii) viable cells.

[0108] Because the causes of low back pain may be myriad, and because ofthe significant cost of some HAAMMPs, it would be useful for a clinicianto first perform a diagnostic test in order to confirm that the targeteddisc in fact possesses high levels of the targeted MMPs prior toproviding the injection.

[0109] In one embodiment, the diagnostic test comprises a non-invasivediagnostic test comprising, for example, using an MRI. In someembodiments, the MRI is able to quantify the aggrecans levels within thedisc.

[0110] In one embodiment, the clinician would first perform a discogramin order to identify which disc or discs are responsible for thepatient's low back pain. Next, the clinician would perform an invasiveor non-invasive test upon the targeted disc in order to confirm thepresence of or quantify the level of the MMPs.

[0111] It is further believed that the present invention can also beused to prevent degeneration of an intervertebral disc in a humanindividual, namely, by following a procedure comprising:

[0112] a) determining a genetic profile of the individual;

[0113] b) comparing the profile of the individual against apre-determined genetic profile level of at-risk humans;

[0114] c) determining that the individual is an at-risk patient; and

[0115] d) injecting an HAAMMP into a disc of the individual.

[0116] It is further believed that transdiscal administration of aneffective amount of other high specificity antagonists ofpro-inflammatory compounds would also help provide therapy to thepatient having DDD. In many embodiments, the transdiscal administrationis of an effective amount of a high specificity antagonist of an enzyme.

[0117] It is further believed that transdiscal administration of aneffective amount of a high specificity antagonist of p38 kinase wouldalso help provide therapy to the patient having DDD. It is believed thatthe p38 kinase site regulates the production of TNF-α, IL-1 and COX-2enzyme. Therefore, in accordance with another embodiment of the presentinvention, there is provided a method of treating degenerative discdisease in an intervertebral disc having a nucleus pulposus, comprisingtransdiscally administering an effective amount of a formulationcomprising a high affinity antagonist of p 38 kinase into anintervertebral disc.

[0118] Some high specificity antagonists of p 38 kinase are disclosed inZhang, J Biol. Chem., 272(20), May 16, 1997 (13397-402); Pargellis,Nature Structural Biology, 9(4), April 2002268-272, and Chae, Bone,28(1), 45-53 (January 2001), and in U.S. Pat. No. 6,541,477(“Goehring”), the specification of which is hereby incorporated byreference in its entirety.

[0119] It is further believed that transdiscal administration of aneffective amount of a high specificity antagonist of the COX-2 enzymewould also help provide therapy to the patient having DDD. It isbelieved that the COX-2 enzyme is a regulator of the production ofprostaglandins, which are involved both in inflammation and thegeneration of pain. Therefore, in accordance with another embodiment ofthe present invention, there is provided a method of treatingdegenerative disc disease in an intervertebral disc having a nucleuspulposus, comprising transdiscally administering an effective amount ofa formulation comprising a high affinity antagonist of COX-2 enzyme intoan intervertebral disc.

[0120] Typical high specificity antagonists of the COX-2 enzyme includeCelecoxib (Searle), Rofecoxib (Merck); Meloxican (Boehringer Manheim);Nimesulide; diclofenac and Lodine.

[0121] It is further believed that transdiscal administration of aneffective amount of a high specificity antagonist of the PLA₂ enzymewould also help provide therapy to the patient having DDD. It isbelieved that the PLA₂ enzyme is a regulator of the production ofprostaglandin. Therefore, in accordance with another embodiment of thepresent invention, there is provided a method of treating degenerativedisc disease in an intervertebral disc having a nucleus pulposus,comprising transdiscally administering an effective amount of aformulation comprising a high affinity antagonist of PLA₂ enzyme into anintervertebral disc. In some embodiments, the high specificityantagonist of PLA2 may be administered systemically.

[0122] At least one high specificity antagonist of PLA₂ is disclosed inKawakami, Clin. Orthop., 351: 241-51 (1998).

[0123] It is further believed that transdiscal administration of aneffective amount of a high specificity antagonist of the NO synthaseenzyme would also help provide therapy to the patient having DDD. It isbelieved that the NO synthase enzyme regulates the production of NO,which is known to have pro-inflammatory effects. Therefore, inaccordance with another embodiment of the present invention, there isprovided a method of treating degenerative disc disease in anintervertebral disc having a nucleus pulposus, comprising transdiscallyadministering an effective amount of a formulation comprising a highaffinity antagonist of NO synthase into an intervertebral disc. In someembodiments, the high specificity antagonists of NO synthase may beadministered systemically.

[0124] Some high specificity antagonists of NO synthase areN-iminoethyl-L-lysine (L-NIL), and N^(G)-monomethyl-L-arginine.

[0125] It is further believed that transdiscal administration of aneffective amount of a high specificity anti-oxidant would also helpprovide therapy to the patient having DDD. It is believed that oxidantsdegrade the nucleus pulposus extra-cellular matrix. Typicalanti-oxidants include free radical scavengers and superoxide dismutaseenzymes. Therefore, in accordance with another embodiment of the presentinvention, there is provided a method of treating degenerative discdisease in an intervertebral disc having a nucleus pulposus, comprisingtransdiscally administering an effective amount of a formulationcomprising a high affinity antioxidant into an intervertebral disc. Insome embodiments, the high specificity antioxidants may be administeredsystemically.

EXAMPLE I

[0126] This non-limiting prophetic example describes how totransdiscally administer a formulation comprising an HAAMMP and salineinto a nucleus pulposus of a degenerating disc.

[0127] First, a clinician uses a diagnostic test to verify that aparticular disc within a patient has high levels of MMPs.

[0128] Next, the clinician provides a local anesthetic (such as 5 mllidocaine) to the region dorsal of the disc of concern to reducesubcutaneous pain.

[0129] Next, the clinician punctures the skin of the patient dorsal thedisc of concern with a relatively large (e.g., 18-19 gauge) needlehaving a stylet therein, and advances the needle through subcutaneousfat and dorsal sacrolumbar ligament and muscles to the outer edge of theintervertebral disc.

[0130] Next, the stylet is removed from the needle.

[0131] Next, the clinician receives a syringe having a smaller gaugeneedle adapted to fit within the larger gauge needle. This needle istypically a 22 or 24 gauge needle. The barrel of the syringe containsthe formulation of the present invention.

[0132] The formulation contains an HAAMMP, and has an HAAMMPconcentration of between about 30 mg/ml and about 60 mg/ml.

[0133] Next, the physician advances the smaller needle co-axiallythrough the larger needle and past the distal end of the larger needle,thereby puncturing the annulus fibrosus. The smaller needle is thenfurther advanced into the center of the nucleus pulposus. Finally, theclinician depresses the plunger of the syringe, thereby injectingbetween about 0.1 and 1 ml of the formulation into the nucleus pulposus.

EXAMPLE II

[0134] This non-limiting prophetic example is substantially similar tothat of Example I, except that the formulation comprises a sustainedrelease device comprising the co-polyer poly-DL-lactide-co-glycolide(PLG). The formulation contains HAAMMP as the antagonist, and has anHAAMMP concentration of between about 30 mg/ml and about 60 mg/ml.

What is claimed is:
 1. A method of treating degenerative disc disease inan intervertebral disc having a nucleus pulposus, comprisingtransdiscally administering an effective amount of a formulationcomprising a high affinity anti-matrix metalloproteinase (HAAMMP) intoan intravertebral disc.
 2. The method of claim 1 wherein the HAAMMP isadministered in an amount effective to inhibit MMPs present in thenucleus pulposus and help arrest degradation of an extracellular matrix.3. The method of claim 1 wherein the HAAMMP is a TIMP.
 4. The method ofclaim 3 wherein the TIMP is a recombinant TIMP.
 5. The method of claim 3wherein the TIMP is present in an autologous concentrated form.
 6. Themethod of claim 1 wherein the HAAMMP is an anti-aggrecanase.
 7. Themethod of claim 6 wherein the anti-aggrecanase is present in anautologous concentrated form.
 8. The method of claim 6 wherein theanti-aggrecanase is a recombinant anti-aggrecanase.
 9. The method ofclaim 1 wherein the formulation is administered in a volume of between0.03 ml and 0.3 ml.
 10. The method of claim 2 wherein the concentrationof HAAMMP in the formulation is at least 100 mg/ml.
 11. The method ofclaim 1 wherein the formulation further comprises at least oneadditional therapeutic agent.
 12. The method of claim 1 wherein theformulation is administered in an amount of less than 1 cc.
 13. Themethod of claim 12 wherein the concentration of HAAMMP in theformulation is at least 100 mg/ml.
 14. The method of claim 1 wherein theformulation further comprises a sustained release device.
 15. The methodof claim 14 wherein the sustained release device comprises a hydrogel.16. The method of claim 14 wherein the sustained release device providescontrolled release.
 17. The method of claim 14 wherein the sustainedrelease device provides continuous release.
 18. The method of claim 14wherein the sustained release device provides intermittent release. 19.The method of claim 14 wherein the sustained release device comprises abiosensor.
 20. The method of claim 14 wherein the sustained releasedevice comprises microspheres.
 21. The method of claim 14 wherein theHAAMMP is predominantly released from the sustained delivery device bydiffusion through the sustained delivery device or by biodegradation ofthe sustained delivery device.
 22. The method of claim 14 wherein thesustained release device comprises an inflammatory-responsive deliverysystem.
 23. The method of claim 1 wherein the HAAMMP is present in theformulation in a maximum amount of about 0.5 mg.
 24. The method of claim1 wherein the formulation further comprises a growth factor present inan amount effective to repair disc tissue.
 25. The method of claim 24wherein the growth factor is a TGF-β.
 26. The method of claim 24 whereinthe growth factor is provided by platelet concentrate.
 27. The method ofclaim 1 wherein the formulation further comprises viable mesenchymalstem cells.
 28. The method of claim 1 wherein the formulation isinjected into the nucleus pulposus.
 29. The method of claim 1 whereinthe formulation is injected into the annulus fibrosus.
 30. The method ofclaim 1 wherein a portion of the nucleus pulposus is removed prior toadministering the formulation into the intervertebral disc.
 31. Themethod of claim 1 wherein the adminstration is performed through aneedle.
 32. The method of claim 31 wherein the needle has a maximumgauge of 24 gauge.
 33. The method of claim 1 wherein the administrationcomprises providing the formulation in a patch attached to an outer wallof the annulus fibrosus.
 34. The method of claim 1 wherein theadministration comprises providing the formulation in a depot closelyadjacent to an outer wall of the annulus fibrosus.
 35. The method ofclaim 1 wherein the adminstration comprises providing the formulation ina depot closely adjacent an endplate of an adjacent vertebral body. 36.The method of claim 1 wherein the degenerating disc is an intact disc.37. The method of claim 1 wherein the degenerating disc is a ruptureddisc.
 38. The method of claim 1 wherein the degenerating disc isdelaminated.
 39. The method of claim 1 wherein the degenerating disc hasfissures.
 40. The method of claim 1 wherein the HAAMMP is predominatlyreleased from the sustained delivery device by diffusion of the HAAMMPthrough a sustained delivery device.
 41. The method of claim 40 whereinthe sustained delivery device is a polymer.
 42. the method of claim 1wherein the HAAMMP is predominantly relased from the sustained deliverydevice by biodegradation of the sustained delivery device.
 43. Themethod of claim 1 wherein the HAAMMP is a specific antagonist of acollagenase MMP.
 44. The method of claim 1 wherein the HAAMP is aspecific anagonist of stromelysin MMP.
 45. The method of claim 1 whereinthe HAAMMP is a specific antagonist of gelatinase MMP.
 46. The method ofclaim 1 wherein the HAAMP is a specific antagonist of a membrane MMP.47. The method of claim 1 wherein the HAAMMP is a specific antagonist ofan MMP selected from the group consisting MMP-2, MMP-3 and MMP-8. 48.The method of claim 1 wherein the HAAMMP is a specific antagonist ofMMP-2.
 49. the method of claim 1 wherein the HAAMMP is a specificantagonist of MMP-3.
 50. The method of claim 1 wherein the HAAMMP is aspecific anatagonist of MMP-8.
 51. A formulation for treatingdegenerative disc disease, comprising: a) a high affinity anti-matrixmethalloproteinase HAAMMP; and b) an additional therapeutic agentselected from the group consisting of: i) a growth factor, and ii)viable mesenchymal stem cells.
 52. the formulation of claim 51 whereinthe additional therapeutic agent is viable mesenchymal stem cells. 53.The formulation of claim 51 wherein the additional therapeutic agent isa growth factor.
 54. The formulation of claim 53 wherein the mesenchymalstem cells are autologous.
 55. the formulation of claim 54 wherein themesenchymal stem cells are provided in a concentrated from.
 56. Theformulation of claim 54 whrein the HAAMMP is selected from the groupconsisting of TIMP-1 and TIMP-2.
 57. The formulation of claim 54 wherinthe HAAMMP is an anti-aggrecanase.
 58. A method of preventingdegeneration of an intervertebral disc in a human individual,comprising; a) determining a genetic profile of individual; b) comparingthe profile of the individual aainst a pre-determined genetic profilelevel of at-risk humans; c) determining that te individual is an at-riskpatient; and d) injecing a high affinity antagonist of MMPs into a discof the individual.